The present invention relates to the gene and peptide sequences of a diabetes-specific endogenous retrovirus derived from type 1 diabetes patients. In particular, the present invention relates to the whole genome of the diabetes-specific variant of endogenous retrovirus (ERV-9) purified from pancreatic tissues of type 1 diabetes (insulin-dependent diabetes mellitus [IDDM]) patients, and viral genes and peptides sequences which can be used to develop a diagnosing reagent for type 1 diabetes and an immunogen.
It is known that the human genome contains a complex variety of inherited endogenous retroviral sequences, several of which are transcriptionally active and contain open reading frames. Aberrant expression of endogenous retroviral sequences has been implicated in the pathogenesis of many autoimmune diseases, including Sjogren""s syndrome (Hao W., et al., J. Autoimmune, 6: 787-798, 1993), rheumatoid arthritis (Garry R. F., Arthritis and Rheumatism, 37: 465-469, 1994), systemic lupus erythematosus (Wilder R. L., Current Opinions in Rheumatology, 6: 295-299, 1994), Hashimoto""s thyroiditis (Blomberg J., et al., Arthritis and Rheumatism. 37: 57-66, 1994), Graves"" disease (Blomberg J., et al., Arthritis and Rheumatism. 37: 57-66, 1994) and the like. In addition, some infectious exogenous retroviruses cause manifestations of autoimmunity (Tomer Y., et al., Endocrine Reviews, 14: 107-120, 1993).
Type 1 diabetes, also known as insulin-dependent diabetes mellitus, is an autoimmune diseases resulting from the destruction of pancreatic beta cells by beta cell-specific autoimmune processes. However, the processes which trigger the autoimmunity remain unknown. In the pancreatic beta cells of non-obese diabetic (NOD) mice, a spontaneously diabetic animal model for human type 1 diabetes, the presence of retrovirus particles in pancreatic beta cells, which is known to be associated with insulitis and diabetes, has been reported. The initiation of insulitis in NOD mice is known to occur at 4 to 6 weeks of age. Fujita et al. observed retrovirus particles in NOD mice as young as 2 days old using electron microscopy (Fujita H., et al., Biochemical Research, 5(1): 71-76, 1984). In addition, Serreze et al. found that anti-type C retrovirus antibody shows peak titer shortly after weaning in NOD mice (Serreze D. V., et al., Diabetes, 37: 351-358, 1988). In this study, type C retrovirus particles were found in both intact and lymphocyte-infiltrated islets, and retrovirus particles were also detected in beta cells showing severe nuclear damage within intact islets which showed no evidence of lymphocytic infiltration. In another study, Like and Rossini observed the induction of aberrant retrovirus budding into the rough endoplasmic reticulum of the beta cells, two to three days before insulitis developed in male CD-1 mice treated with multiple doses of streptozotocin (Like A. A. and Rossini A. A., Science, 193: 415-417, 1976). These results suggest that the expression of retrovirus occurs prior to lymphocytic infiltration of the islets. Thus, the expression of retrovirus has been assumed to be an initial event in the damage of beta cells, rather than the result of insulitis.
To date, there has been no report of the exclusive expression of retrovirus particles in the beta cells of recent-onset type 1 diabetes patients. Thus, an examination on whether retrovirus particles are expressed in pancreatic tissues of type 1 diabetes patients was required to clarify the etiology of the onset of autoimmune type 1 diabetes.
Therefore, the present inventors have extensively examined the expression of retrovirus particles in pancreatic tissues of type 1 diabetes patients, and have found that retrovirus particles are expressed specifically in the pancreatic beta cells of diabetes patients. This fact can be exploited to develop a diagnosing reagent for type 1 diabetes and an immunogen. In particular 21 domains deduced from the above retroviral gene were elucidated to develop new peptides for vaccines and the like because these domains show hydrophilicity and immuno-dominancy.
The present inventors have also elucidated that the diabetes-specific retrovirus is an endogenous retrovirus (ERV-9) variant.
Although a direct relationship between the onset of type 1 diabetes and the expression of diabetes-specific endogenous retrovirus (ERV-9) was found, the role of beta cell-specific expression of retroviruses in the pathogenesis of beta cell-specific autoimmunity remains unknown. There are three possible mechanisms whereby retrovirus may be involved in the etiology of the disease.
First, the presentation of a retroviral antigen of the beta cells by MHC class II molecules from antigen-presenting cells (APCs) , such as macrophages, may be the initial step in the autoimmune destruction of beta cells (Chomczynski P., et al., Anal. Biochem., 162: 156, 1987; Froussard P. A., Nucleic Acids Research, 20(11): 2900, 1992; Shin A., et al., J. virol., 63(1): 64-75, 1989). An immune response to a specific antigen on a target cell involves the activation of CD4+ T cells, which are activated only when they interact with antigens presented on the surface of a macrophage or other APC.
Second, retroviral genome in the beta cells may alter the expression of cellular genes, possibly resulting in a beta cell-specific altered antigen(s) which could be recognized as foreign by the immune system. The retroviral antigen or altered beta cell antigen might be presented by MHC class I molecules and recognized as foreign by CD8+ cytotoxic T cells (CTLs). Subsequently, signals transduced through T cell receptors (TcR) may be activated and CTLs may produce cytokines such as interferon-xcex3(INF-xcex3) which would, in turn, induce inflammation and further stimulate antigen presentation, leading to beta cell-specific autoimmunity (York I. A. and Rock K. L., Annu. Rev. Immunol., 14: 369-396, 1996).
Third, retroviral antigen-specific effector T cells, which may recognize autoantigens expressed on the beta cells by molecular mimicry, may be generated.
The object of the present invention is to provide a whole genome of a diabetes-specific endogenous retrovirus (ERV-9) derived from the pancreatic tissues of type 1 diabetes patients, including the gag gene, pol gene, and env gene of the viral genome and their nucleotide sequences, shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, respectively.
The object of the present invention is to provide amino acid sequences deduced from the above gene sequences. In particular, the Gag protein has the amino acid sequence shown in SEQ ID NO:3, the Pol protein has the amino acid sequence shown in SEQ ID NO:5 and the Env gene has the amino acid sequence shown in SEQ ID NO:7.
The object of the present invention is to provide antigens of the diabetes-specific endogenous retrovirus (ERV-9) containing the entire or partial amino acid sequence deduced from the above gag, pol or env gene sequences.
The object of the present invention is to provide antibodies for the diabetes-specific endogenous retrovirus (ERV-9), prepared by using the above antigens.
The object of the present invention is to provide vaccines for the diabetes-specific endogenous retrovirus (ERV-9) containing the above antigens as effective agents.
The object of the present invention is to provide diagnosing reagents for diabetes, which are prepared by using the above nucleotide sequences entirely and partially.
The object of the present invention is to provide diagnosing reagents for diabetes, which are prepared by using the entire or partial peptide sequences derived from Gag, Pol or Env protein.
The object of the present invention is to provide diagnosing reagents for diabetes, which are prepared by exploiting the above antibodies.
The object of the present invention is to provide a peptide and its derivative, which comprises the amino acid sequence containing the antigenic determinant or immuno-dominant domain deduced from the gene sequence of the diabetes-specific endogenous retrovirus (ERV-9).
The object of the present invention is to provide diagnosing reagents for diabetes, which contain the above peptide as an effective agent.
The object of the present invention is to provide antibodies for the diabetes-specific endogenous retrovirus (ERV-9), which are prepared by using the above peptide.
The object of the present invention is to provide vaccines for diseases related to the diabetes-specific endogenous retrovirus (ERV-9), which contain the above peptide as an effective agent.